Project summary Reversible phosphorylation of proteins modulates their function and thereby regulates virtually all cellular processes. Whereas phosphorylation of serine, threonine and tyrosine are exceedingly well characterized, relatively little is known about phosphorylation of histidine, which may account for as much as ~6% of all incorporation of phosphate into mammalian proteins. The identity of the kinases and phosphatases that regulate histidine phosphorylation, their protein targets, and their biological functions have remained obscure. Over the past several years, we provided genetic and biochemical evidence that the histidine kinase, nucleoside diphosphate-B (NDPK-B), and the histidine phosphatase, protein histidine phosphatase 1 (PHPT1) regulate the activity of the Ca2+-activated K+ channel KCa3.1 and, therefore the activation of CD4 T cells and mast. These discoveries likely represent only the ?tip of the iceberg? and serve as the foundation for future work because it is likely that many other pathways are regulated by histidine phosphorylation. We have now identified only the second histidine phosphatase, which specifically histidine dephosphorylates and inhibits NDPK-B and, via this effect, is a potent negative regulator KCa3.1 and CD4 T cells. Moreover, using recently developed monoclonal antibodies to 1- and 3-phospho-Histidine (pHis), we demonstrate for the first time the regulation of histidine phosphorylation in vivo in mammalian cells, which we in turn linked to TCR signaling. Our proposal will address the regulation, protein targets, and biologic roles for this newly identified histidine phosphatase, which will then be extended to general properties of histidine phosphorylation in regulating biological processes in mammalian cells. In Aim 1, we will identify the mechanism(s) whereby this newly identified histidine phosphates is regulated and in turn regulates NDPK-B dephosphorylation, modulates activation of downstream pathways, and the role of this histidine phosphatase as a histidine phosphatase for other pHis proteins. In addition, using recently developed anti-1- and 3-pHis antibodies, coupled with other preliminary data generated, we propose a novel strategy to identify new histidine phosphorylated proteins. In Aim 2, we will determine the role and regulation of this newly identified histidine phosphatase to suppress immune cell signaling. We found that siRNA knockdown of the histidine phosphatase in human Th0 CD4 cells and Th0 CD4 cells isolated from knockout mice have increased 1-pHis phosphorylation of NDPK-B, leading to increased activation of KCa3.1 and a subsequent increase in TCR-stimulated Ca2+ flux and cytokine production. We will explore the regulation and function(s) of this phosphatase in the context of TCR signaling, its role in specific CD4 T cell subsets, and whether it functions in vivo to limit autoimmune disease. We will also will extend findings in TCR signaling in CD4 T cells to Fc?R1-stimulated activation of mast cells.